Summary of Work: This proposal seeks to define the mechanism(s) by which E. coli MutS and MutL block recombination between closely related but non-identical DNA. At present studies have primarily focused on the role of MutS and its associated ATPase activity. In replication fidelity MutS initiates repair by binding the mismatch. The exact function of MutL is not known. Current models suggest MutL adds to the MutS-mismatch complex in an ATP-dependent fashion. MutS has within its c-terminal domain both Walker A & B consensus sequences. The exact role of ATP binding/hydrolysis in methyl-directed repair is not known but has stimulated great interest because of the internal conservation of MutS in higher cells. We now know that nucleotide binding by E.coli, yeast, and human MutS results in loss of mismatch recognition. Consequently it is highly probable that ATP hydrolysis is the driving force behind MutS-dependent DNA translocation along the DNA helix contour. To help clarify the biochemistry behind MutS ATPase activity we have carried out site-directed mutagenesis to define the active site residue (carboxylate) responsible for hydrolysis. We have preliminary evidence to suggest aspartic acid 661 is the active site residue. Currently, we are characterizing several other residues that might participate in hydrolysis and testing their ability to bind a G/T mismatch. Previously our lab investigated the dominant negative effect associated with two MutS mutants in vitro. We were, however, unable to dissect the genetics behind this dominant negative phenotype in vivo with our given biochemical strategies. Therefore we decided to tackle this problem utilizing an approach that would allow us to prepare heterodimers between mutant and wild type MutS. This procedure involved differentially GST- and his6- fusion proteins. The separate tags facilitated rapid and pure 1:1 mutant-wild-type heterodimers. However, this procedure was met with failure in that the GST-fusion was not active. We therefore constructed a different tag utilizing the affinity of strepavidin for strepavidin binding peptide. Preliminary studies suggest a 1:1 ratio of mutant to wild type hinders repair but does not completely block it. We are currently testing the possibility that MutS bidirectional repair capacity is a function of independent ATP-driven monomer translocation. In examining further roles of mismatch repair in recombination we demonstrate that MutS & MutL proteins block RuvAB-dependent branch migration between M13-fd DNA. This effect was mismatch dependent, as branched intermediates between M13-M13 were unimpeded by these activities.